Tumor Budding Correlates With the Protumor Immune Microenvironment and Is an Independent Prognostic Factor for Recurrence of Stage I Lung Adenocarcinoma

Abstract

Background: Immune cell infiltration associated with tumor capsule disruption and tumor budding has been shown to reflect invasiveness, metastasis, and unfavorable prognosis in colorectal cancer. We investigated the influence of tumor budding on prognosis and its association with the immune microenvironment in lung adenocarcinoma.

Methods: Tumor slides from resected stage I lung adenocarcinomas were reviewed (n = 524 and n = 514, for training and validation cohorts, respectively) for assessment of tumor budding. CD3+ and forkhead box P3+ (FoxP3+) lymphocytes, CD68+ macrophages, IL-7 receptor, and IL-12 receptor β2 were analyzed using tissue microarrays constructed from tumor and stroma. Probability of recurrence was calculated using the competing risks method.

Results: In the training cohort, risk of recurrence for high-grade tumor budding was higher than it was for low-grade tumor budding (32% vs 12%, P < .001), which was confirmed in the validation cohort (P = .005). Tumor budding stratified the risk of recurrence for acinar-predominant (22% vs 9%, P < .001), papillary-predominant (22% vs 13%, P = .045), and solid-predominant (39% vs 19%, P = .022) tumors. Tumor budding was associated with higher stromal FoxP3+ lymphocyte infiltration, higher stromal FoxP3/CD3 risk index, higher tumoral and stromal CD68+ macrophage infiltration, and IL-7 receptor overexpression (P < .001, all associations). Tumor budding remained independently associated with recurrence on multivariate analysis (hazard ratio, 1.61; P = .008).

Conclusions: Tumor budding is an independent prognostic factor of stage I lung adenocarcinoma and correlates with the protumor immune microenvironment. Our findings advocate investigating tumor-immune cell interactions at the invading edge as a biologic driver of tumor aggressiveness.

Figure 1 –

Flowchart of the study design. We reviewed patients with pathologic stage I lung adenocarcinoma who had undergone surgical resection (N = 1,038). Tumors were classified according to the IASLC/ATS/ERS classification. Prognostic immune scores (CD3, FoxP3, CD68, IL-7R, and IL12-Rβ2), based on tissue microarray analysis, were dichotomized as low or high. To dichotomize tumor budding as low or high, the entire cohort was split into a training cohort (n = 524) and a validation cohort (n = 514). After reviewing tumor slides at intermediate-power fields at ×100 magnification, tumor budding was assessed at the area with the maximal number of smallest tumor nests composed of fewer than five tumor cells and was quantified by counting 10 HPFs at ×200 magnification. During evaluation using 10 HPFs, maximum number of tumor buds per one HPF was considered the number of buds for each tumor. Tumor budding was classified as grade 0 (zero buds per HPF), grade 1 (one to four), grade 2 (five to nine), or grade 3 (≥ 10). On the basis of our results, tumors were classified as having high-grade (grade 2-3) or low-grade (grade 0-1) tumor budding. FoxP3 = forkhead box P3; HPF = high-power field; IASLC/ATS/ERS = International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society; IL-7R = IL-7 receptor; IL-12Rβ2 = IL-12 receptor β2.

Figure 2 –

A-D, Histologic findings of tumor budding (original magnifications: A, ×40; B, ×400; C, D, ×200). A, Tumor budding identified in stroma of the invasive tumor edge (circle). B, Tumor budding defined as isolated small tumor nests composed of fewer than five tumor cells (arrows). C, Low-grade tumor budding. D, High-grade tumor budding.

Figure 3 –

A, B, CIR analysis of tumor budding. A, In the training cohort, 5-y CIR for patients with high-grade tumor budding was significantly higher (32%) than it was for patients with low-grade budding (12%; P < .001). B, In the validation cohort, 5-y CIR for patients with high-grade tumor budding was significantly higher (20%) than it was for patients with low-grade budding (12%; P = .005). CIR = cumulative incidence of recurrence.

Figure 4 –

A-D, Subgroup analysis of CIR analysis of tumor budding by stage and surgical procedure. A, In patients with stage IA disease, 5-y CIR for patients with high-grade tumor budding was significantly higher (19%) than it was for patients with low-grade budding (10%; P = .003). B, In patients with stage IB disease, 5-y CIR for patients with high-grade tumor budding was significantly higher (34%) than it was for patients with low-grade budding (17%; P < .001). C, In patients undergoing lobectomy, 5-y CIR for patients with high-grade tumor budding was significantly higher (22%) than it was for patients with low-grade budding (10%; P < .001). (D) In patients undergoing limited resection, 5-y CIR for patients with high-grade tumor budding was significantly higher (45%) than it was for patients with low-grade budding (18%; P < .001). See Figure 3 legend for expansion of abbreviation.

Figure 5 –

Association between tumor budding and histologic subtype. High-grade tumor budding was most frequently identified in solid-predominant tumors (53%), followed by micropapillary-predominant (42%), and acinar-predominant (34%) tumors. Rate of high-grade tumor budding was significantly higher in solid-predominant tumors than in tumors of other subtypes (P < .001). AIS = adenocarcinoma in situ; MIA = minimally invasive adenocarcinoma.